FITOTE-02959; No of Pages 11 Fitoterapia xxx (2014) xxx–xxx

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Fitoterapia

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journal homepage: www.elsevier.com/locate/fitote

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Jian Chen, Hong Wu ⁎, Miao-Miao Dai, Hui Li, Jin-Yun Chen, Shun-Li Hu

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College of Pharmacy, Anhui University of Chinese Medicine, Key Laboratory of Modernized Chinese Medicine in Anhui Province, Hefei, Anhui, China

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Identification and distribution of four metabolites of geniposide in rats with adjuvant arthritis

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Keywords: Geniposide Metabolites Information-Dependent Acquisition (IDA) LC–MS/MS Adjuvant arthritis

Geniposide (GE), also called Jasminoidin, is the major active ingredient of Gardenia jasminoides Ellis (GJ) fruit, which has long been used in traditional Chinese medicine (TCM). Growing evidences suggested that GE has a great potentiality for treating rheumatoid arthritis (RA). However, GE is rapidly metabolized, and we know little about its availability or metabolites in tissues. To elucidate the distribution of GE and its metabolites in tissues, three groups of adjuvant arthritis (AA) rats were given GE (33, 66 and 120 mg/kg) from days 18 to 24, and the biotransformation of GE in plasma, liver, spleen, synovium, urine and mesenteric lymph node (MLN) of rats was investigated by a novel approach named Information-Dependent Acquisition (IDA)-Mediated LC–MS/MS method. As a result, GE and its four major metabolites were detected as follows: GE, G1, G2 in plasma; GE, G2 in MLNs; only GE in liver and synovium; GE, G2, G3 and G4 in spleen; and GE, G1, G2 and G4 in urine. In total four metabolites (G1–G4) involved in the in vivo metabolism processes were identified. The results of this work have demonstrated the IDA-Mediated LC–MS/MS could screen rapidly and reliably the characterization of metabolites from iridoid compounds. © 2014 Published by Elsevier B.V.

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Article history: Received 11 March 2014 Accepted in revised form 27 May 2014

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1. Introduction

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RA is a chronic systemic disease of unknown etiology, which is characterized by an inflammatory process in synovium resulting in progressive destruction of cartilage and bone in

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Abbreviations: GE, geniposide; TCM, traditional Chinese medicine; RA, rheumatoid arthritis; MLN, mesenteric lymph node; AA, adjuvant arthritis; IDA, Information-Dependent Acquisition; SRM, selected reaction monitoring; ESI, electrospray ionization; EPI, enhanced product ion; SD, Sprague– Dawley; FCA, Freund's complete adjuvant; Ep, Eppendorf; MRM, the multiple reaction monitoring; TIC, total ion chromatogram; DP, declustering potential; CE, collision energies; CXP, Cell Exit Potential; XIC, extracted ion chromatogram; EP, Entrance Potential; CUR, Curtain Gas; IS, IonSpray Voltage; TEM, Temperature; Gas 1, Ion Source Gas 1; Gas 2, Ion Source Gas 2; GlucA, glucuronic acid; PK, pharmocokinetics. ⁎ Corresponding author at: College of Pharmacy, Anhui University of Chinese Medicine, Hefei 230031, China. Tel.: +86 551 6516 9230; fax: +86 551 6516 9222. E-mail address: [email protected] (H. Wu).

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affected joints [1–3]. Moreover, it is the most common inflammatory arthritis and a major cause of disability [4]. GE (Fig. 1(A)), also called Jasminoidin, is a water-soluble iridous glycoside purified from gardenia fruit which has been reported to treat hepatic and inflammatory conditions [5–9]. However, recent reports [10,11] suggested that GE causes liver toxicity, and most of the investigators believed that the transformation process of GE into aglycone genipin (Fig. 1(B)) or other metabolites was related to the liver toxicity of GE. Therefore, it is important to find out, which of the protagonist for the pharmacological effects of GE is, itself or its metabolites. AA is widely used as an experimental model, which shares some features with human RA in some pathological, histological and immunological aspects [12]. Some studies have focused on the quantification of GE in normal rat plasma and the identification of metabolites in normal rat urine with LC– MS/MS [13–15]. Our preliminary results showed that the time of GE reaching Cmax (peak concentration) was at 1 h after oral administration with GE in AA rat plasma [16]. Han et al. found

http://dx.doi.org/10.1016/j.fitote.2014.05.023 0367-326X/© 2014 Published by Elsevier B.V.

Please cite this article as: Chen J, et al, Identification and distribution of four metabolites of geniposide in rats with adjuvant arthritis, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.05.023

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Fig. 1. Chemical structure of geniposide (A), genipin (B), metabolite G1 (C), metabolite G2 (D), metabolite G3 (E), metabolite G4 (F).

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2. Experimental

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2.1. Materials

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GE (purity N 98%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The standard of genipin (purity N 98%) was supplied by Chengdu Must Bio-technology Co., Ltd. LC grade formic acid was purchased from ROE. Acetonitrile and methanol, HPLC grade, were obtained from Fisher (Shanghai, China).

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that the glucuronidation of the aglycone of GE and its ring-cleavage derivatives was the main metabolic products observed in urine sample [15]. Moreover, it was proved that GE would transform into genipin and a nitrogen-containing metabolite genipinine by intestinal bacteria in humans [17]. However, further researches in specific disease state and associated areas involving immune tissues and effector tissues have not been reported. It is helpful for further revealing the pharmacological mechanism of GE and screening new therapeutic formulas for RA to explore the metabolism of GE in immune tissues of rats with AA. Information-Dependent Acquisition (IDA)-Mediated LC–MS/MS method is an advanced approach in qualitative study of many target compounds and has been successfully used to support plasma pharmacokinetic screening programs [18]. This method presents a screening procedure based on the injection of tissue samples (after handled) in a LC–ESI-MS/MS system (AB SCIEX QTRAP® 4500). Its feasibility within a forensic toxicological setting has been demonstrated by assessing the potential and pitfalls of the IDA-Mediated LC–MS/MS screening approach [19].

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Deionized water was prepared by Milli-Q Ultrapure water purification system (Millipore, Bedford, MA, USA). Physiological saline samples were purchased from the Tian Bao Biopharmaceutical., Ltd. Guangdong Province (brand A, plastic bag).

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2.2. Apparatus

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Instrumentation-MS/MS analysis was constitute of an Agilent 1290 binary pump HPLC system (Agilent technologies Inc., USA) fitted with an ACQUITY UPLC™ HSS C18 column (100 mm × 2.1 mm i.d., 1.8 μm particle size) interfaced to an AB SCIEX Triple Quad TM 4500 Mass Spectrometers (AB SCIEX, USA). An Analyst® 1.6.1 Software controlled the LC–ESI-MS/MS system and processed the data. Centrifuge (Eppendorf 5430R, Germany) was purchased from Eppendorf.

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2.3. IDA-Mediated LC–MS/MS condition

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2.3.1. Chromatographic conditions The mobile phase consisted of 0.1% formic acid in water (A) and 90% formic acid in acetonitrile (B). The gradient applied was as follows: from 0 to 2 min 98% A and 2% B, from 2 to 10 min to 100% B, from 10 to 11 min to 98% A, and from 11 min 98% A and 2% B. Run time was 15 min followed by a 1 min delay prior to the next injection. Flow rate was 0.2 mL/min. Column temperature was kept at 20 °C.

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2.3.2. Mass spectral conditions and IDA Mass detection is performed in the negative ion IDA mode, a selected reaction monitoring (SRM) experiment as survey scan and the “enhanced product ion” (EPI) scan as dependent

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Please cite this article as: Chen J, et al, Identification and distribution of four metabolites of geniposide in rats with adjuvant arthritis, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.05.023

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From day 18 to 24 after immunization, the low-dosage, middle-dosage and high-dosage groups were given GE intragastrically at 33, 66 and 120 mg/kg (in physiological saline), respectively, once daily, while the AA group and the normal group were given an equal volume of physiological

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Table 1 SRM parameters, retention times and product ions of the studied compounds.

t1:3 Retention time (min) Precursor ions (m/z) SRM Molecular (m/z) CUR (psi) IS (V) CAD (psi) TEM (°C) Gas 1 (psi) Gas 2 (psi) DP (V) EP (V) CXP (V) Scan rate (Da/s) CE ± CES (eV) Product ion

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t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19

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Sprague–Dawley (SD) rats (♂, 180 ± 20 g. Grade II, Certificate No. 011) were purchased from the Animal Department of Anhui University of Chinese Medicine (Hefei, China) and acclimatized to our animal house for at least 3 days in advance to the experiments. All the experiments were performed in accordance with Guidelines for animal Experiments of Anhui University of Chinese Medicine and the rats were euthanized under anesthesia condition. Rats were randomly divided into five groups, namely, high-dosage group, middle-dosage group, low-dosage group, AA group and normal group, and there were six rats in each group. AA rats were induced by a single intradermal injection into the left hind metatarsal footpad with 100 μL of Freund's complete adjuvant (FCA) for each rat, and the normal group was induced with 100 μL physiological saline instead. Rats were examined daily for signs of arthritis by two independent observers who were not aware of the treatment. Non-injected hind paw volume was determined with YLS-7A volume meter (Shandong Academy of Medical Sciences Equipment Station, China). Paw swelling (mL) was graded on a scale of 0–4: 0, no swelling; 1, isolated swelling of finger joints; 2, involvement of ankle or wrist joints; 3, severe inflammation of the entire paws; and 4, involvement of entire paw, including ankle. The maximum joint score was 12 including three secondary arthritis paws for each rat.

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varied within a specified range, and the resulting spectrum is: 150 CE − CES, CE, and CE + CES. 151

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acquisition [20]. When the collected signal strength of a SRM channel exceeds a preset value (i.e. “peak”), the system will automatically switch (b1 ms) to the linear ion trap mode, trigger EPI scan and obtain the MS/MS spectra of the precursor ions that corresponds to SRM channel. The SRM experiment was performed at unit resolution at both quadrupoles, with a dwell time of 75 ms. MS parameters, including declustering potential (DP), Collision Energy (CE), Entrance Potential (EP), and Cell Exit Potential (CXP) were studied for GE and genipin by infusing in the spectrometer a 1 μg/mL solution in water separately (Table 1). The Source/Gas parameters including Curtain Gas (CUR), IonSpray Voltage (IS), Temperature (TEM), Ion Source Gas 1 (Gas 1) and Ion Source Gas 2 (Gas 2) were optimized by FIA compound optimization in negative MS/MS mode (224.900/122.600 and 224.9/101.000 for genipin, 386.900/122.600 and 386.900/224.900 for GE) by infusing 1 μg/mL genipin and GE solution mixed in blank tissues. IDA parameters included the acquisition of a minimum of 1 and up to 2 ions whose peak height exceeded 500 counts and the exclusion for 60 s after 5 acquisitions of the same ion. EPI was performed with Q1 set at unit resolution and the triple–quadrupole (QqQ) scanning from 50 to 600 amu, with a step size of 0.2 Da (at a rate of 200 Da/s). A single spectrum needed three scans. The parameters were used during the EPI experiment as detailed in Table 1. The IDA software permitted not only the collection of one SRM transition for each compound, but also the acquisition of the EPI spectrum of each substance found in the chromatogram undergoing the selection criteria for the acquisition of the dependent scan. However, one flaw of an IDA procedure was that it could not optimize the fragmentation parameters for each different precursor during the product ion scan. The use of a CE common to all compounds might preclude all chance of reaching a good fragmentation, especially if very different chemical structures are involved, as in the case of the group metabolites of GE. In order to resolve this problem, the collision energy spread (CES) may be applied, so that CE is

Geniposide

Genipin

G1

G2

G3

G4

5.12 225, 122.9 386.9/122.6 or 224.9/122.6 432.9, 387.0 30 −4500 9 400 75 65 −30 −10 −10 200 20 ± 5 224.7, 122.6, 44.9

5.27 225, 122.9 224.9/122.6 224.9 30 −4500 9 400 75 65 −25 −10 −10 200 20 ± 5 206.8, 146.4, 122.5, 100.0

5.19 225, 122.9 400.9/122.6 401.1 30 −4500 9 400 75 65 −40 −10 −10 200 25 ± 5 383, 368.6, 224.4, 206.8, 122.7, 100.7

5.27 225, 122.9 224.9/122.6 225.2 30 −4500 9 400 75 65 −25 −10 −10 200 20 ± 5 206.7, 147.0, 122.5, 100.0

3.97 225 224.9/122.6 375.7 30 −4500 9 400 75 65 −100 −10 −10 200 25 ± 5 332.8, 224.9, 206.5, 149.7, 132.6, 110.6

3.87 225 224.9/122.6 359.7 30 −4500 9 400 75 65 −100 −10 −10 200 25 ± 5 317.0, 224.0, 208.4, 149.8, 133.8, 110.6

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Please cite this article as: Chen J, et al, Identification and distribution of four metabolites of geniposide in rats with adjuvant arthritis, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.05.023

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3.1. Collection of plasma samples

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A 0.5 mL aliquot of blood sample was collected from the venous plexus of eye at 0, 1, 1.5, and 5 h after a single dose by oral administration of GE to rats. The blood samples were centrifuged at 5000 × g for 10 min and stored at 20 °C until further analysis.

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3.2. Collection of urine samples

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Five groups (high-dosage group, middle-dosage group, low-dosage group, AA group and normal group) were fasted for 12 h with water ad libitum prior to the administration of GE. Then the low-dosage, middle-dosage and high-dosage groups were given GE intragastrically at 33, 66 and 120 mg/kg (in physiological saline), respectively, while the AA group and the normal group were given an equal volume of physiological saline at the same time. The liver and spleen samples were collected at 1, 1.5, and 5 h. The liver and spleen samples were perfused and washed with ice-cold buffer (10 mM potassium phosphate, pH 7.4).

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Six rats (high-dosage group) were held in metabolic cages and kept in environmentally controlled breeding room with standard laboratory food and water for one week prior to the 196 experiments. All animals were fasted for 12 h with water ad 197 libitum prior to the administration of GE. During the time, blank 198 urine samples were collected. Then the rats were administered 199 at a dose of 120 mg/kg GE in saline. Finally, urine samples 200 collected at 0, 2, 4, 6, 8, and 24 h post-dosing in an ice bath, 201 Q12 were immediately transferred to a deep freezer and were 202 stored at −20 °C until analysis.

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The assay standard samples were prepared by spiking 200 μL or 0.2 g dry-weight AA group samples with 500 μL GE or genipin (1 μg/mL) to extract for 0.5 min, then centrifuged at 22,000 ×g (4 °C) for 10 min and de-proteinized with 500 μL ice-cold methanol. Solvents contained in the extract obtained after centrifugation were evaporated under N2 and the residue was dissolved in 500 μL acetonitrile/water/formic acid (50: 50: 0.1, v/v/v).

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3.3. Collection of liver, spleen, MLN and synovium samples

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saline at the same time. GE was dissolved in water to give three final concentrations of 3.3, 6.6, and 12 mg/mL for oral administration.

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Fig. 2. Analysis in negative ionization mode of geniposide at 1 mg/L in water: extracted ion chromatogram (XIC) of −MRM (m/z 386.9/122.6) detected in negative ionization (A, peak at 5.11 min: geniposide standard), EPI spectrum of the peak at 5.11 min (B, precursor: m/z 386.9).

Please cite this article as: Chen J, et al, Identification and distribution of four metabolites of geniposide in rats with adjuvant arthritis, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.05.023

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500 μL acetonitrile/water/formic acid (50: 50: 0.1, v/v/v), vortex 242 mixed, centrifuged at 22,000 ×g (4 °C) for 10 min and 2 μL 243 supernatant was injected into the LC–MS system for analysis. 244

4.1. Preparation of plasma sample

200 μL aliquot of plasma sample was collected into Eppendorf (Ep) tubes at 0, 1, 1.5 and 5 h after a single dose 227 by oral administration of GE to rats (n = 30). Mixed with 228 500 μL of methanol and vortexed for 30 s then centrifuged at 229 5000 ×g (4 °C) for 10 min. Transferred supernatant into 230 another Ep tube and evaporated the eluent to dryness by a 231 gentle stream of nitrogen at 37 °C, finally the residues were 232 reconstituted in 500 μL acetonitrile/water/formic acid (50: 233 50: 0.1, v/v/v) vortex mixed, centrifuged at 22,000 × g (4 °C) 234 Q14 for 10 min. Each sample (2 μL) was injected into LC–MS 235 system for analysis.

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1 mL urine sample was vortex-mixed with 4 mL methanol to extraction for 0.5 min, and then centrifuged at 5000 ×g (4 °C) for 10 min to separate precipitated proteins. The supernatant was separated and evaporated to dryness under a stream of N2 gas at room temperature. The residue was reconstituted in

5. Validation

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Considering the fact that the procedure was developed for 257 screening purposes, validation was limited to the verification of 258

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Fresh rat livers, spleens, MLNs and synoviums were harvested from euthanized rats, weighed and minced. Minced livers were homogenized using a motorized homogenizer in three-time volume of ice-cold homogenization buffer (10 mM potassium phosphate, pH 7.4) and centrifuged again at 5000 ×g (4 °C) for 10 min, the supernatant was collected and mixed with 1 mL methanol then centrifuged again at 22,000 ×g (4 °C) for 10 min. The supernatant obtained after centrifugation was dried by a gentle stream of nitrogen at 37 °C and the extracts were treated as plasma.

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Fig. 3. Analysis in negative ionization mode of genipin at 1 μg/mL in water: Extracted ion chromatogram (XIC) of −MRM (m/z 224.9/122.6) detected in negative ionization (A, peak at 5.30 min: geniposide standard), EPI spectrum of the peak at 5.27 min (B, precursor: m/z 224.9).

Please cite this article as: Chen J, et al, Identification and distribution of four metabolites of geniposide in rats with adjuvant arthritis, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.05.023

J. Chen et al. / Fitoterapia xxx (2014) xxx–xxx

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Please cite this article as: Chen J, et al, Identification and distribution of four metabolites of geniposide in rats with adjuvant arthritis, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.05.023

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Fig. 5. MS/MS spectrum of the [M − H]− ion at m/z 401.1 for metabolite G1 (A); MS/MS spectrum of [M − H]− ion at m/z 225.2 for metabolite G2 (B) from drug-containing plasma.

method selectivity (6 different blanks of each tissue injected), to the determination of the limits of detection (LOD) and to the investigation of matrix effects. The limit of detections (LODs) of GE and genipin were determined as a signal-to-noise ratio of 3:1. The blank tissues with different concentrations (0, 0.5, 1.0, 2.5, 5.0 ng/mL) of spiked GE and genipin were independently prepared and determined, using an optimized UPLC condition and MS tune parameters.

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Matrix effects were assessed by comparing the peak areas of GE after adding high (1000 ng/mL), medium (100 ng/mL), and low concentrations (10 ng mL) of GE and genipin to the mobile phase (A) and the supernatant of extracted blank tissues (B). These studies were conducted with six different rat tissues. The peak area ratio of B/A (as a percentage) was used as a quantitative measure for the matrix effect and ion suppression.

Fig. 4. XICs of −MRM after an overlay in vivo metabolism of GE: (A) plasma (A1, blank plasma; A2, plasma sample obtained 1 h after oral administration of GE at a dose of 120 mg/kg); (B) liver (B1, blank liver; B2, liver sample obtained 1 h after oral administration of GE at a dose of 120 mg/kg); (C) spleen (C1, blank spleen; C2, spleen sample obtained 2 h after oral administration of GE at a dose of 120 mg/kg); (D) MLNs (D1, blank MLNs; D2, MLN sample obtained 1 h after oral administration of GE at a dose of 120 mg/kg); (E) synovium (E1, blank synovium; E2, synovium sample obtained 1 h after oral administration of GE at a dose of 120 mg/kg); (F) urine (F1, blank urine; F2, urine sample obtained 1 h after oral administration of GE at a dose of 120 mg/kg). (1, GE; G1, metabolite 1; G2, metabolite; G3, metabolite; G4, metabolite).

Please cite this article as: Chen J, et al, Identification and distribution of four metabolites of geniposide in rats with adjuvant arthritis, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.05.023

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The intraday assay precision was obtained by analyzing GE and genipin of B samples six times on the same day.

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6. Results and discussion

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The retention times of GE and its metabolites under the reported chromatographic conditions are listed in Table 1. Negative ionization SRM chromatograms of GE and genipin at 1 mg/L in water are shown in Figs. 2 and 3, respectively.

spectra of [M + HCOOH-H]− of GE standard (Fig. 2(B)), and MS/MS spectra with the authentic reference sample (Table 1). In addition, to directly identify genipin (aglycone of GE), UPLC retention time (Fig. 3(A)) and MS/MS (Fig. 3(B)) data were acquired. It can be seen from the MS/MS spectra that GE and genipin share the same product ions at m/z 224.7, 206.8, 122.6, and 100.0. And the different dissociation product ions were selected as the precursor ions for subsequent MS/MS experiment to provide structural information in the negative ion mode.

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6.1. Parent compound and its aglycone

6.2. Metabolite G1

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The non-metabolized GE was identified by comparing the UPLC retention time (Fig. 2(A)), the representative MS/MS

The metabolite G1 (Fig. 1(C)) showed an UPLC profile with 295 a retention time at 5.19 min and an ESI spectrum which gave a 296

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Fig. 6. MS/MS spectrum of the [M − H]− ion at m/z 359.7 for metabolite G4 (A); MS/MS spectrum of [M − H]− ion at m/z 375.7 for metabolite G3 (B) from drug-containing plasma.

Please cite this article as: Chen J, et al, Identification and distribution of four metabolites of geniposide in rats with adjuvant arthritis, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.05.023

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t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14

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Plasma Liver Spleen MLNs Synovium Urine

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Matrix effects (B/A, %)

LODs

10 ng/mL

100 ng/mL

1000 ng/mL

95.6 95.8 91.7 96.8 94.6 99.4

98.7 92.4 103.0 93.8 100. 2 99.3

101.2 102.7 98.4 98.2 101.8 100.6

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6.3. Metabolite G2

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Metabolite G2 (Fig. 1(D)) with a retention time of 5.27 min produced a molecular ion at m/z 225.2 (Fig. 4(A2)). Product ion at m/z 206.7 was produced by a loss of H2O from molecular ion. The MS/MS spectrum of the precursor ion at m/z 225 and 122.9 extracted from G2 showed the main product ions at m/z 206.7, 147.0, 122.5 and 100.0 (Fig. 5(B)), which were identical to those obtained from genipin (Fig. 3(B)). G2 was identified as genipin, which is in agreement with the MS data and retention time of the reference standard.

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6.4. Metabolites G3 and G4

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100 ng/mL

1000 ng/mL

98.9 98.8 98.7 96.5 98.4 105.2

100.8 96.5 98.4 99.0 101.2 93.9

99.3 100.2 98.8 101.0 98.6 95.3

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6.5. Distribution of GE and its metabolites

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10 ng/mL

The limit of detections (LODs, signal-to-noise ratio = 3:1) 337 for GE (Rt: 5.12 min) and genipin (Rt: 5.27 min) in matrix 338 (blank tissue) are listed in Table 2. In the present study, all ratios 339 of B/A were between 91.7 and 105.2, suggesting that the matrix 340 effect or ion suppression for GE and genipin could be ignored. 341 The coefficient of variability was less than 3.3% from intra-assay 342 showing that the repeatability of the system was good (Table 3). 343 As shown in XIC of MRM after an overlay in vivo metabolism 344 of GE (Fig. 4), GE and its four major metabolites were detected as 345 follows: GE, G1, G2 in plasma; GE, G2 in MLNs; only GE in liver 346 and synovium; GE, G2, G3 and G4 in spleen; and GE, G1, G2 and 347 G4 in urine. 348 Fig. 7 shows that the peak areas of GE and its metabolites 349 varied in the AA rat plasma (Fig. 7(A): 224.9/122.6, G2; 401.0/ 350 122.6, G1; 386.9/122.6, GE), liver (Fig. 7(B): 386.9/122.6, GE), 351 spleen (Fig. 7(C): 386.9/122.6, GE; 224.9/122.6, Rt 5.27, G2; 352 224.9/122.6, Rt 3.97, G3; 224.9/122.6, Rt 3.87, G4), MLNs 353 (Fig. 7(D): 386.9/122.6, GE; 224.9/122.6, G2), synovium 354 (Fig. 7(E): 386.9/122.6, GE) and urine (Fig. 7(F): 401.0/122.6, 355 G1; 224.9/122.6, G2; 386.9/122.6, GE; 375.1/122.6, G3) of 356 middle-dosage at the monitoring time-points. We could initially 357 infer pharmacokinetic characteristics of GE and its metabolites 358 in AA rat tissues from peak areas to GE and each metabolite after 359

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molecular ion at m/z 401.1 (Fig. 4(A2)). The fragment ion of G1 was similar to that of GE, and the fragment ions at m/z 383.1, 369.0, 224.6, 174.4 and 122.5 were detected. The loss of 174.4 Da indicates that G1 is a glucuronide conjugate and GE aglycone at m/z 224.6 confirms that the structure of this metabolite contained the aglycone (genipin) of GE (Fig. 5(A)). Hence, it was identified as the mono-glucuronide conjugate of genipin and it has been reported in the urine of rats [15].

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Genipin (ng/mL)

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GE (ng/mL)

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Tissue

D

t2:6

Table 2 Limits of detection (LOD), determined as a signal-to-noise ratio of 3:1, and matrix effect to GE and genipin, assessed by comparing the peak areas of GE after adding high (1000 ng/mL), medium (100 ng/mL), and low concentrations (10 ng mL) of GE and genipin to the mobile phase (A) and the supernatant of extracted blank tissues (B). These studies were conducted with six different rat tissues. The peak area ratio of B/A (as a percentage) was used as a quantitative measure of the matrix effect and ion suppression.

E

t2:1 t2:2 t2:3 t2:4 t2:5

9

N C

317 Q16 Metabolites G3 (Fig. 1(E)) and G4 (Fig. 1(F)) were eluted at 318 the retention times of 3.97 and 3.87 min, respectively. Metab319 Q17 olite G3 produced a molecular ion at m/z 375.7 and metabolite 320 G4 produced a molecular ion at m/z 359.7 (Fig. 4(C2)). The

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MS/MS spectrum obtained from G4 displayed the same product ions as the fragment ions of metabolite G3 (Fig. 6(A) and (B)), and suggesting that the metabolite G4 could be a loss of O from G3. In addition, the diagnostic losses of a molecule of CO2 (44 Da) were observed in the MS/MS spectra of each metabolite and the product ion at 332.8 of G3 was also 15.8 Da higher than the product ion at m/z 317.0 of G4. As shown in MS/MS spectral data (Fig. 6(A)), the molecular ion at m/z 359.7 lost a molecule of CO2 to generate a product ion at m/z 317.0. It displayed similar product ions with GE, such as 224.0, 208.4, and 149.8, but smaller fragments were different from those of GE. In addition, the product ion at m/z 133.8 could be a loss of CH4 from a cysteine. Hence, we could predict that G4 would be a cysteine conjugate ring-opened genipin and G3 could be an oxidation of G4.

U

321 322

Table 3 Intraassay variability of the peak areas of GE and genipin in AA rat tissues.

t3:3

Precision (C.V., %)

Plasma Liver Spleen MLNs Synovium Urine

GE Genipin GE GE Genipin GE Genipin GE GE Genipin

t3:1 t3:2

10 ng/mL

100 ng/mL

1000 ng/mL

t3:4

3.2 1.0 3.3 3.2 1.0 3.3 3.0 1.6 2.0 1.1

2.7 1.4 0.78 1.9 1.7 1.6 0.18 1.7 1.8 0.54

1.6 0.41 2.0 1.0 0.38 0.67 0.27 1.0 1.0 0.62

t3:5 t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12 t3:13 t3:14

Please cite this article as: Chen J, et al, Identification and distribution of four metabolites of geniposide in rats with adjuvant arthritis, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.05.023

J. Chen et al. / Fitoterapia xxx (2014) xxx–xxx

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oral administration of depurated GE at 66 mg/kg in AA rats as follows: First, genipin (aglycone of GE) could be detected in plasma, spleen, MLNs and urine. Recent studies [15,21] about the metabolism and PK of genipin and GE in rats, found that genipin could not be detected after oral administration of GE. GE would decompose into genipin in gastrointestinal tract, and then quickly combined a number of small molecules such as glucuronic acid after oral administration [11,15]. In addition, Hou [21] found that the parent form of genipin declined very rapidly and could not be detected in rat plasma later than 60 min post dosing by HPLC method. The rapid conjugation with amino acids might also in part contribute to the attenuation of genipin in plasma [22,23]. Our results revealed that genipin could be detected in

U

360

N

C

Fig. 7. Mean peak area-time profile of geniposide (GE) and metabolites after oral administration of 66 mg/kg GE in rat. A (in plasma: 224.9/122.6, G2; 401.0/122.6, G1; 386.9/122.6, GE), B (in liver: 386.9/122.6, GE), C (in spleen: 386.9/122.6, GE; 224.9/122.6, Rt 5.27, G2; 224.9/122.6, Rt 3.97, G3; 224.9/122.6, Rt 3.87, G4), D (in MLNs: 386.9/122.6, GE; 224.9/122.6, G2), E (in synovium: 386.9/122.6, GE), F (in urine: 401.0/122.6, G1; 224.9/122.6, G2; 386.9/122.6, GE; 375.1/122.6, G3).

plasma, spleen, MLNs and urine. Therefore, the results of this work have demonstrated that the IDA-Mediated LC–MS/MS could screen rapidly and reliably the characterization of metabolites from iridoid compounds. Second, in rat liver could merely be detected GE, we speculate that GE could not be broken down or transformed by liver enzymes when GE is orally administered. Recent studies have suggested that the low and variable bioavailability of GE could be attributed to the poor absorption or undergoing multiple-stage and unpredictable metabolism from the biological matrix [15]. Akao et al. [24] found that subcellular fractions did not transform GE into any metabolite and speculated that GE could not be broken down or transformed by liver enzymes. However, GE would be decomposed into active aglycones with human

Please cite this article as: Chen J, et al, Identification and distribution of four metabolites of geniposide in rats with adjuvant arthritis, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.05.023

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7. Conclusion

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In this paper a simple and sensitive LC–MS method was developed for the determination of GE and relevant metabolites in rat. In addition, metabolites seem to be effectively produced in the intestine and then absorbed to act as anti-inflammatory genuine when GE is orally administered. In total four metabolites (G1–G4) involved in the in vivo metabolism processes were identified. GE itself could be detected in synovium. The results of this work have demonstrated that the IDA-Mediated LC–MS/ MS could screen rapidly and reliably the characterization of metabolites from iridoid compounds.

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We gratefully thank the Experimental Center of Anhui University of Chinese Medicine. This work was financially supported by National Natural Science Foundation of China (81073122), Anhui Provincial Natural Science Research Project of University (KJ2009A045Z) and the youth Natural Science Foundation of Anhui University of Chinese Medicine (qn201308).

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intestinal function [24,25]. Therefore, GE and other metabolites seem to be effectively produced in the intestine and absorbed to act as genuine anti-inflammatories. However, GE could be detected in the synovium with no other metabolites. It reveals that GE itself could be its anti-inflammatory immunity active ingredient. Hence, further experiments on the cellular and molecular level should be performed to explain the possible anti-inflammatory mechanism. Third, in total four metabolites (G1–G4) involved in the in vivo metabolism processes were identified. Next we would focus more on the pharmacokinetics, bioactivity and anti-inflammatory of the metabolites.

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Please cite this article as: Chen J, et al, Identification and distribution of four metabolites of geniposide in rats with adjuvant arthritis, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.05.023

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Identification and distribution of four metabolites of geniposide in rats with adjuvant arthritis.

Geniposide (GE), also called Jasminoidin, is the major active ingredient of Gardenia jasminoides Ellis (GJ) fruit, which has long been used in traditi...
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